A new field of biomedical research, biomechanics of hemostasis and thrombosis, has been quickly developing over the past few years. The mechanical properties of fibrin are naturally variable and largely determine whether clots stanch bleeding, or lead to thrombosis or hemorrhage, and this makes them a desirable therapeutic target. In this application, fibrin mechanics will be studied with respect to structural changes during physiologically relevant fibrin deformations at increasing levels of complexity, including individual molecules, fibrin oligomers, and whole fibrin clots as well as ex vivo thrombi. The structural basis of the viscoelastic properties of fibrin is going to be examined using a uniquely broad, integrated approach based on state-of-the- art biophysical techniques, such as single-molecule optical trap-based force spectroscopy, wide angle X-ray scattering, Fourier Transform infrared spectroscopy, high-resolution rheometry, atomic force microscopy, confocal and electron microscopy, combined with computational molecular dynamics simulations and multiscale modeling. In Specific Aim 1, the structural transitions in fibrin at the molecular level induced by mechanical force will be studied. Understanding of the unfolding of the coiled-coils, ?C regions, and ?-nodules will define the molecular changes that occur in vivo as a result of blood flow, clot retraction, and wound stretching. The ?-helix to ?-sheet transition in the coiled-coils is an important mechanism of fibrin mechanics and potentially tunable for clinical purposes. Straightening of the ?C polymers and unfolding of the ?-nodules also play major roles in fibrin mechanical properties. In Specific Aim 2, nanomechanics of the A:a knob-hole bonds that hold fibrin together will be studied at the single-molecule level. Preliminary data show that at the A:a bonds exhibit counterintuitive catch bond behavior, meaning that the strength of the bond increases with increasing force. This novel finding is a basis for further in depth studies because of its general importance for the field of biomolecular interactions and potential physiological significance. Using a new approach, Binding- Unbinding Correlation Spectroscopy, that we developed we will extensively characterize the two-dimensional kinetics and thermodynamics of formation and dissociation of single A:a bonds. In Specific Aim 3, mechanical properties of clinically significant clots and thrombi will be studied, with a logical progression from the molecular and microscopic levels to increasingly complex macroscopic structures formed in vivo. Screening of chemicals and structural modifications that potentially stabilize or destabilize fibrin molecular domains will be performed to reveal potential modulators of fibrin mechanical properties for therapeutic purposes. These studies would advance the field of hemostasis and thrombosis by leading to new structure- and mechanics-based approaches to prevent and treat bleeding and thrombosis.